Unrestrained markerless trait stacking in Nannochloropsis gaditana through combined genome editing and marker recycling technologies.

Robust molecular tool kits in model and industrial microalgae are key to efficient targeted manipulation of endogenous and foreign genes in the nuclear genome for basic research and, as importantly, for the development of algal strains to produce renewable products such as biofuels. While Cas9-mediated gene knockout has been demonstrated in a small number of algal species with varying efficiency, the ability to stack traits or generate knockout mutations in two or more loci are often severely limited by selectable agent availability. This poses a critical hurdle in developing production strains, which require stacking of multiple traits, or in probing functionally redundant gene families. Here, we combine Cas9 genome editing with an inducible Cre recombinase in the industrial alga Nannochloropsis gaditana to generate a strain, NgCas9+Cre+, in which the potentially unlimited stacking of knockouts and addition of new genes is readily achievable. Cre-mediated marker recycling is first demonstrated in the removal of the selectable marker and GFP reporter transgenes associated with the Cas9/Cre construct in NgCas9+Cre+ Next, we show the proof-of-concept generation of a markerless knockout in a gene encoding an acyl-CoA oxidase (Aco1), as well as the markerless recapitulation of a 2-kb insert in the ZnCys gene 5'-UTR, which results in a doubling of wild-type lipid productivity. Finally, through an industrially oriented process, we generate mutants that exhibit up to ∼50% reduction in photosynthetic antennae size by markerless knockout of seven genes in the large light-harvesting complex gene family.

Lipid production in Nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator.

Lipid production in the industrial microalga Nannochloropsis gaditana exceeds that of model algal species and can be maximized by nutrient starvation in batch culture. However, starvation halts growth, thereby decreasing productivity. Efforts to engineer N. gaditana strains that can accumulate biomass and overproduce lipids have previously met with little success. We identified 20 transcription factors as putative negative regulators of lipid production by using RNA-seq analysis of N. gaditana during nitrogen deprivation. Application of a CRISPR-Cas9 reverse-genetics pipeline enabled insertional mutagenesis of 18 of these 20 transcription factors. Knocking out a homolog of fungal Zn(II)2Cys6-encoding genes improved partitioning of total carbon to lipids from 20% (wild type) to 40-55% (mutant) in nutrient-replete conditions. Knockout mutants grew poorly, but attenuation of Zn(II)2Cys6 expression yielded strains producing twice as much lipid (∼5.0 g m-2 d-1) as that in the wild type (∼2.5 g m-2 d-1) under semicontinuous growth conditions and had little effect on growth.

The protein Compromised Hydrolysis of Triacylglycerols 7 (CHT7) acts as a repressor of cellular quiescence in Chlamydomonas.

Microalgae are prolific photosynthetic organisms that have the potential to sustainably produce high-value chemical feedstocks. However, an industry based on microalgal biomass still is faced with challenges. For example, microalgae tend to accumulate valuable compounds, such as triacylglycerols, only under stress conditions that limit growth. To investigate the fundamental mechanisms at the base of this conundrum--the inverse relationship between biomass production and storage compound accumulation-we applied a combination of cell biological and genetic approaches. Conceptually, nutrient deprivation, which commonly is used to induce the accumulation of triacylglycerol in microalgae, leads to a state of cellular quiescence defined by a halt of cell divisions that is reversible upon nutrient resupply. To identify factors that govern cellular quiescence, we screened for mutants of the model alga Chlamydomonas reinhardtii that, in contrast to wild-type cells placed under conditions of nitrogen deprivation, were unable to degrade triacylglycerols following nitrogen resupply. One of the mutants described here in detail, compromised hydrolysis of triacylglycerols 7 (cht7), was severely impaired in regrowth following removal of different conditions inducing cellular quiescence. The mutant carries a deletion affecting four genes, only one of which rescued the quiescence phenotype when reintroduced. It encodes a protein with similarity to mammalian and plant DNA binding proteins. Comparison of transcriptomes indicated a partial derepression of quiescence-related transcriptional programs in the mutant under conditions favorable to growth. Thus, CHT7 likely is a repressor of cellular quiescence and provides a possible target for the engineering of high-biomass/high-triacylglycerol microalgae.

A galactoglycerolipid lipase is required for triacylglycerol accumulation and survival following nitrogen deprivation in Chlamydomonas reinhardtii.

Following N deprivation, microalgae accumulate triacylglycerols (TAGs). To gain mechanistic insights into this phenomenon, we identified mutants with reduced TAG content following N deprivation in the model alga Chlamydomonas reinhardtii. In one of the mutants, the disruption of a galactoglycerolipid lipase-encoding gene, designated PLASTID GALACTOGLYCEROLIPID DEGRADATION1 (PGD1), was responsible for the primary phenotype: reduced TAG content, altered TAG composition, and reduced galactoglycerolipid turnover. The recombinant PGD1 protein, which was purified from Escherichia coli extracts, hydrolyzed monogalactosyldiacylglycerol into its lyso-lipid derivative. In vivo pulse-chase labeling identified galactoglycerolipid pools as a major source of fatty acids esterified in TAGs following N deprivation. Moreover, the fatty acid flux from plastid lipids to TAG was decreased in the pgd1 mutant. Apparently, de novo-synthesized fatty acids in Chlamydomonas reinhardtii are, at least partially, first incorporated into plastid lipids before they enter TAG synthesis. As a secondary effect, the pgd1 mutant exhibited a loss of viability following N deprivation, which could be avoided by blocking photosynthetic electron transport. Thus, the pgd1 mutant provides evidence for an important biological function of TAG synthesis following N deprivation, namely, relieving a detrimental overreduction of the photosynthetic electron transport chain.

Galactoglycerolipid metabolism under stress: a time for remodeling.

Galactoglycerolipids are the predominant lipid building blocks of chloroplast membranes and are essential for plant growth. Plant chloroplasts harbor a constitutive set of UDP-Gal-dependent lipid galactosyltransferases that are responsible for the bulk of galactoglycerolipid biosynthesis. A set of paralogs is induced in response to phosphate deprivation, which leads to the remodeling of extraplastidic membranes with a partial replacement of phosphoglycerolipid by digalactosyldiacylglycerol. A third type of galactoglycerolipid biosynthetic enzyme, a UDP-Gal-independent galactoglycerolipid galactosyltransferase, was recently shown to be involved in freezing tolerance. Here, we look at how understanding of the regulation of galactoglycerolipid biosynthesis in chloroplasts by these multiple enzyme sets is rapidly evolving and discuss the increasingly recognized role of lipid remodeling in response to diverse abiotic stresses.

Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism.

Like many microalgae, Chlamydomonas reinhardtii forms lipid droplets rich in triacylglycerols when nutrient deprived. To begin studying the mechanisms underlying this process, nitrogen (N) deprivation was used to induce triacylglycerol accumulation and changes in developmental programs such as gametogenesis. Comparative global analysis of transcripts under induced and noninduced conditions was applied as a first approach to studying molecular changes that promote or accompany triacylglycerol accumulation in cells encountering a new nutrient environment. Towards this goal, high-throughput sequencing technology was employed to generate large numbers of expressed sequence tags of eight biologically independent libraries, four for each condition, N replete and N deprived, allowing a statistically sound comparison of expression levels under the two tested conditions. As expected, N deprivation activated a subset of control genes involved in gametogenesis while down-regulating protein biosynthesis. Genes for components of photosynthesis were also down-regulated, with the exception of the PSBS gene. N deprivation led to a marked redirection of metabolism: the primary carbon source, acetate, was no longer converted to cell building blocks by the glyoxylate cycle and gluconeogenesis but funneled directly into fatty acid biosynthesis. Additional fatty acids may be produced by membrane remodeling, a process that is suggested by the changes observed in transcript abundance of putative lipase genes. Inferences on metabolism based on transcriptional analysis are indirect, but biochemical experiments supported some of these deductions. The data provided here represent a rich source for the exploration of the mechanism of oil accumulation in microalgae.

Freezing tolerance in plants requires lipid remodeling at the outer chloroplast membrane.

Plants show complex adaptations to freezing that prevent cell damage caused by cellular dehydration. Lipid remodeling of cell membranes during dehydration is one critical mechanism countering loss of membrane integrity and cell death. SENSITIVE TO FREEZING 2 (SFR2), a gene essential for freezing tolerance in Arabidopsis, encodes a galactolipid remodeling enzyme of the outer chloroplast envelope membrane. SFR2 processively transfers galactosyl residues from the abundant monogalactolipid to different galactolipid acceptors, forming oligogalactolipids and diacylglycerol, which is further converted to triacylglycerol. The combined activity of SFR2 and triacylglycerol-biosynthetic enzymes leads to the removal of monogalactolipids from the envelope membrane, changing the ratio of bilayer- to non-bilayer-forming membrane lipids. This SFR2-based mechanism compensates for changes in organelle volume and stabilizes membranes during freezing.

Lipid transport mediated by Arabidopsis TGD proteins is unidirectional from the endoplasmic reticulum to the plastid.

The transfer of lipids between the endoplasmic reticulum (ER) and the plastid in Arabidopsis involves the TRIGALACTOSYLDIACYLGLYCEROL (TGD) proteins. Lipid exchange is thought to be bidirectional based on the presence of specific lipid molecular species in Arabidopsis mutants impaired in the desaturation of fatty acids of membrane lipids in the ER and plastid. However, it was unclear whether TGD proteins were required for lipid trafficking in both directions. This question was addressed through the analysis of double mutants of tgd1-1 or tgd4-3 in genetic mutant backgrounds leading to a defect in lipid fatty acid desaturation either in the ER (fad2) or the plastid (fad6). The fad6 tgd1-1 and fad6 tgd4-3 double mutants showed drastic reductions in the relative levels of polyunsaturated fatty acids and of galactolipids. The growth of these plants and the development of photosynthetic membrane systems were severely compromised, suggesting a disruption in the import of polyunsaturated fatty acid-containing lipid species from the ER. Furthermore, a forward-genetic screen in the tgd1-2 dgd1 mutant background led to the isolation of a new fad6-2 allele with a marked reduction in the amount of digalactosyldiacylglycerol. In contrast, the introduction of fad2, affecting fatty acid desaturation of lipids in the ER, into the two tgd mutant backgrounds did not further decrease the level of fatty acid desaturation in lipids of extraplastidic membranes. These results suggest that the role of TGD proteins is limited to plastid lipid import, but does not extend to lipid export from the plastid to extraplastidic membranes.

Phosphate regulation of lipid biosynthesis in Arabidopsis is independent of the mitochondrial outer membrane DGS1 complex.

Galactoglycerolipids are major constituents of photosynthetic membranes in chloroplasts. At least three parallel sets of enzymes are involved in their biosynthesis that must be coordinated in response to changing growth conditions. A potential candidate for a protein affecting the activity of different galactoglycerolipid pathways is the recently described digalactosyldiacylglycerol1 (dgd1) SUPPRESSOR1 (DGS1) protein of Arabidopsis (Arabidopsis thaliana) localized in the mitochondrial outer membrane. It was discovered based on a specific gain-of-function point mutation allele, dgs1-1, that causes a partial restoration of chloroplast galactoglycerolipid deficiency in the dgd1 mutant, which is defective in the lipid galactosyltransferase, DGD1. The dgs1-1 allele causes the accumulation of hydrogen peroxide that leads to an activation of an alternative, DGD1-independent galactoglycerolipid biosynthesis pathway in chloroplasts. Analysis presented here shows that the DGS1 protein is a component of a large protein complex, which explains the previously observed dominant negative phenotype following the expression of the dgs1-1 allele. The dgs1-1 allele causes the loss of mitochondrial alternative oxidase (AOX) protein that might be related to the accumulation of hydrogen peroxide in the dgs1-1 mutant background. This effect was posttranscriptional because mRNA levels for the major form of AOX were not affected in dgs1-1 mutant seedlings. Unlike dgs1-1, a loss-of-function allele, dgs1-2, had no effect on plant growth, AOX, and lipid composition to the extent tested, leaving the quest for a possible molecular function of DGS1 open. Apparently, the DGS1 wild-type protein does not directly affect lipid metabolism in mitochondria or chloroplasts.

RNA interference silencing of a major lipid droplet protein affects lipid droplet size in Chlamydomonas reinhardtii.

Eukaryotic cells store oils in the chemical form of triacylglycerols in distinct organelles, often called lipid droplets. These dynamic storage compartments have been intensely studied in the context of human health and also in plants as a source of vegetable oils for human consumption and for chemical or biofuel feedstocks. Many microalgae accumulate oils, particularly under conditions limiting to growth, and thus have gained renewed attention as a potentially sustainable feedstock for biofuel production. However, little is currently known at the cellular or molecular levels with regard to oil accumulation in microalgae, and the structural proteins and enzymes involved in the biogenesis, maintenance, and degradation of algal oil storage compartments are not well studied. Focusing on the model green alga Chlamydomonas reinhardtii, the accumulation of triacylglycerols and the formation of lipid droplets during nitrogen deprivation were investigated. Mass spectrometry identified 259 proteins in a lipid droplet-enriched fraction, among them a major protein, tentatively designated major lipid droplet protein (MLDP). This protein is specific to the green algal lineage of photosynthetic organisms. Repression of MLDP gene expression using an RNA interference approach led to increased lipid droplet size, but no change in triacylglycerol content or metabolism was observed.

Mutation of a mitochondrial outer membrane protein affects chloroplast lipid biosynthesis.

Lipid biosynthesis in plant cells is associated with various organelles, and maintenance of cell lipid homeostasis requires nimble regulation and coordination. In plants, environmental cues such as phosphate limitation require readjustment of the lipid biosynthetic machinery to substitute phospholipids by non-phosphorous glycolipids. Biosynthesis of the galactoglycerolipids predominant in plants proceeds by a constitutive and an alternative pathway that is known to be induced in response to phosphate deprivation. Plant lipid galactosyltransferases involved in both pathways are associated with the plastid envelope membranes and are encoded by nuclear genes. To identify mechanisms governing the activity of the alternative galactoglycerolipid pathway, a genetic suppressor screen was conducted in the background of the digalactolipid-deficient dgd1 mutant of Arabidopsis. A suppressor line that partially restored digalactoglycerolipid content in the dgd1 background carries a point mutation in a mitochondrial protein, which was tentatively designated DGD1 SUPPRESSOR 1 (DGS1). Presumed orthologs of this protein are present in plants, algae and fungi, but its molecular function is not yet known. In the dgd1 dgs1 double mutant, expression of nuclear genes encoding enzymes of the alternative galactoglycerolipid pathway is increased and hydrogen peroxide levels are elevated. This increase in hydrogen peroxide is proposed to be the reason for activation of the alternative pathway in the dgd1 dgs1 double mutant. Accordingly, hydrogen peroxide and treatments producing reactive oxygen also activate the alternative pathway in the wild-type. These results likely implicate the production of reactive oxygen in the regulation of the alternative galactoglycerolipid pathway in plants.

The two divergent PEP-carboxylase catalytic subunits in the green microalga Chlamydomonas reinhardtii respond reversibly to inorganic-N supply and co-exist in the high-molecular-mass, hetero-oligomeric Class-2 PEPC complex.

Our recent molecular studies revealed two divergent PEP-carboxylase (PEPC [Ppc]) encoding genes in the green microalga Chlamydomonas reinhardtii, CrPpc1 and CrPpc2, which are coordinately responsive to changes in inorganic-N and -C supply at the transcript level [Mamedov, T.G., Moellering, E.R. and Chollet, R. (2005) Identification and expression analysis of two inorganic C- and N-responsive genes encoding novel and distinct molecular forms of eukaryotic phosphoenolpyruvate carboxylase in the green microalga C. reinhardtii, Plant J. 42, 832-843]. Here, we report the distribution of these two encoded catalytic subunits in the minor Class-1 and predominant Class-2 PEPC enzyme-forms, the latter of which is a novel high-molecular-mass, hetero-oligomeric complex containing both CrPpc1 (p109) and CrPpc2 (p131) polypeptides. The Class-1 enzyme, however, is a typical PEPC homotetramer comprised solely of p109. We also document that the amount of both CrPpc1/2 catalytic subunits is up-/down-regulated by varying levels of NH(4)(+) supplied to the culture medium.

Identification and expression analysis of two inorganic C- and N-responsive genes encoding novel and distinct molecular forms of eukaryotic phosphoenolpyruvate carboxylase in the green microalga Chlamydomonas reinhardtii.

Phosphoenolpyruvate carboxylase (PEPC [Ppc]) has been previously purified and characterized in biochemical and immunological terms from two green microalgae, Chlamydomonas reinhardtii and Selenastrum minutum. The findings indicate that these algae possess at least two distinct PEPC enzyme-forms, homotetrameric Class-1 and heteromeric Class-2, that differ significantly from each other and their plant and prokaryotic counterparts. Surprisingly, however, green-algal PEPC has been unexplored to date in molecular terms. This study reports the molecular cloning of the two Ppc genes in C. reinhardtii (CrPpc1, CrPpc2), each of which is transcribed in vivo and encodes a fully active, recombinant PEPC that lacks the regulatory, N-terminal seryl-phosphorylation domain that typifies the vascular-plant enzyme. These distinct catalytic subunit-types differ with respect to their (i) predicted molecular mass ( approximately 108.9 [CrPpc1] versus approximately 131.2 kDa [CrPpc2]) and critical C-terminal tetrapeptide; and (ii) immunoreactivity with antisera against the p102 and p130 polypeptides of S. minutum PEPC1/PEPC2 and PEPC2, respectively. Only the Ppc1 transcript encodes the p102 catalytic subunits common to both Class-1 and Class-2 enzyme-forms in C. reinhardtii. The steady-state transcript levels of both CrPpc1/2 are coordinately up-/down-regulated by changes in [CO2] or [NH] during growth, and generally mirror the response of cytoplasmic glutamine synthetase (Gs1) transcript abundance to changes in inorganic [N] at 5% CO2. These collective findings provide key molecular insight into the Ppc genes and corresponding PEPC catalytic subunits in the eukaryotic algae.